Nucleic acids encoding heat stable mutants of plant ADP-glucose pyrophosphorylase

ABSTRACT

The subject invention pertains to novel mutant polynucleotide molecules that encode enzymes that have increased heat stability. These polynucleotides, when expressed in plants, result in increased yield in plants grown under conditions of heat stress. The polynucleotide molecules of the subject invention encode maize endosperm ADP glucose pyrophosphorylase (AGP) and soluble starch synthase (SSS) enzyme activities. Plants and plant tissue bred to contain, or transformed with, the mutant polynucleotides, and expressing the polypeptides encoded by the polynucleotides, are also contemplated by the present invention. The subject invention also concerns methods for isolating polynucleotides and polypeptides contemplated within the scope of the invention. Methods for increasing yield in plants grown under conditions of heat stress are also provided.

CROSS-REFERENCE TO A RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/079,478, filed Feb. 19, 2002 now U.S. Pat. No. 6,809,235, which is acontinuation-in-part of U.S. application Ser. No. 09/312,433, filed May14, 1999, now U.S. Pat. No. 6,403,863, which is a continuation-in-partof U.S. application Ser. No. 08/972,545, filed Nov. 18, 1997, now U.S.Pat. No. 6,069,300, and U.S. application Ser. No. 09/312,433 claims thebenefit of U.S. Provisional Application No. 60/085,460, filed May 14,1998, and U.S. application Ser. No. 08/972,545 claims the benefit ofU.S. Provisional Application No. 60/031,045, filed Nov. 18, 1996.

This invention was made with government support under National ScienceFoundation grant number 9316887. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The sessile nature of plant life generates a constant exposure toenvironmental factors that exert positive and negative effects on plantgrowth and development. One of the major impediments facing modemagriculture is adverse environmental conditions. One important factorwhich causes significant crop loss is heat stress. Temperature stressgreatly reduces grain yield in many cereal crops such as maize, wheat,and barley. Yield decreases due to heat stress range from 7 to 35% inthe cereals of world-wide importance.

A number of studies have identified likely physiological consequences ofheat stress. Early work by Hunter et al. (Hunter, R. B., Tollenaar, M.,and Breuer, C. M. [1977] Can. J. Plant Sci. 57:1127-1133) using growthchamber conditions showed that temperature decreased the duration ofgrain filling in maize. Similar results in which the duration of grainfilling was adversely altered by increased temperatures were identifiedby Tollenaar and Bruulsema (Tollenaar, M. and Bruulsema, T. W. [1988]Can. J. Plant Sci. 68:935-940). Badu-Apraku et al. (Badu-Apraku, B.,Hunter, R. B., and Tollenaar, M. [1983] Can. J. Plant. Sci. 63:357-363)measured a marked reduction in the yield of maize plants grown under theday/night temperature regime of 35/15° C. compared to growth in a 25/15°C. temperature regime. Reduced yields due to increased temperatures isalso supported by historical as well as climatological studies(Thompson, L. M. [1986] Agron. J. 78:649-653; Thompson, L. M. [1975]Science 188:535-541; Chang, J. [1981] Agricul. Metero. 24:253-262; andConroy, J. P., Seneweera, S., Basra, A. S., Rogers, G., andNissen-Wooller, B. [1994] Aust. J. Plant Physiol. 21:741-758).

That the physiological processes of the developing seed are adverselyaffected by heat stress is evident from studies using an in vitro kernelculture system (Jones, R. J., Gengenbach, B. G., and Cardwell, V. B.[1981] Crop Science 21:761-766; Jones, R. J., Ouattar, S., andCrookston, R. K. [1984] Crop Science 24:133-137; and Cheikh, N., andJones, R. J. [1995] Physiol. Plant. 95:59-66). Maize kernels cultured atthe above-optimum temperature of 35° C. exhibited a dramatic reductionin weight.

Work with wheat identified the loss of soluble starch synthase (SSS)activity as a hallmark of the wheat endosperm's response to heat stress(Hawker, J. S. and Jenner, C. F. [1993] Aust. J. Plant Physiol.20:197-209; Denyer, K., Hylton, C. M., and Smith, A. M. [1994] Aust. J.Plant Physiol. 21:783-789; Jenner, C. F. [1994] Aust. J. Plant Physiol.21:791-806). Additional studies with SSS of wheat endosperm show that itis heat labile (Rijven, A. H. G. C. [1986] Plant Physiol. 81:448-453;Keeling, P. L., Bacon, P. J., Holt, D. C. [1993] Planta. 191:342-348;Jenner, C. F., Denyer, K., and Guerin, J. [1995] Aust. J. Plant Physiol.22:703-709).

The roles of SSS and ADP glucose pyrophosphorylase (AGP) under heatstress conditions in maize is less clear. (AGP) catalyzes the conversionof ATP and α-glucose-1-phosphate to ADP-glucose and pyrophosphate.ADP-glucose is used as a glycosyl donor in starch biosynthesis by plantsand in glycogen biosynthesis by bacteria. The importance of ADP-glucosepyrophosphorylase as a key enzyme in the regulation of starchbiosynthesis was noted in the study of starch deficient mutants of maize(Zea mays) endosperm (Tsai, C. Y., and Nelson, Jr., O. E. [1966] Science151:341-343; Dickinson, D. B., J. Preiss [1969] Plant Physiol.44:1058-1062).

Ou-Lee and Setter (Ou-Lee, T. and Setter, T. L. [1985] Plant Physiol.79:852-855) examined the effects of temperature on the apical or tipregions of maize ears. With elevated temperatures, AGP activity waslower in apical kernels when compared to basal kernels during the timeof intense starch deposition. In contrast, in kernels developed atnormal temperatures, AGP activity was similar in apical and basalkernels during this period. However, starch synthase activity duringthis period was not differentially affected in apical and basal kernels.Further, heat-treated apical kernels exhibited an increase in starchsynthase activity over control. This was not observed with AGP activity.Singletary et al. (Singletary, G. W., Banisadr, R., and Keeling, P. L.[1993] Plant Physiol. 102:6 (suppl); Singletary, G. W., Banisadra, R.,Keeling, P. L. [1994] Aust. J. Plant Physiol. 21:829-841) using an invitro culture system quantified the effect of various temperaturesduring the grain fill period. Seed weight decreased steadily astemperature increased from 22-36° C. A role for AGP in yield loss isalso supported by work from Duke and Doehlert (Duke, E. R. and Doehlert,D. C. [1996] Environ. Exp. Botany. 36:199-208).

Work by Keeling et al. (1994, supra) quantified SSS activity in maizeand wheat using Q₁₀ analysis, and showed that SSS is an importantcontrol point in the flux of carbon into starch.

In vitro biochemical studies with AGP and SSS clearly show that bothenzymes are heat labile. Maize endosperm AGP loses 96% of its activitywhen heated at 57° C. for five minutes (Hannah, L. C., Tuschall, D. M.,and Mans, R. J. [1980] Genetics 95:961-970). This is in contrast topotato AGP which is fully stable at 70° C. (Sowokinos, J. R. and Preiss,J. [1982] Plant Physiol. 69:1459-1466; Okita, T. W., Nakata, P. A.,Anderson, J. M., Sowokinos, J., Morell, J., and Preiss, J. [1990] PlantPhysiol. 93:785-90). Heat inactivation studies with SSS showed that itis also labile at higher temperatures, and kinetic studies determinedthat the Km value for amylopectin rose exponentially when temperatureincreased from 25-45° C. (Jenner et al., 1995, supra).

Biochemical and genetic evidence has identified AGP as a key enzyme instarch biosynthesis in higher plants and glycogen biosynthesis in E.coli (Preiss, J. and Romeo, T. [1994] Progress in Nuc. Acid Res. and MolBiol. 47:299-329; Preiss, J. and Sivak, M. [1996] “Starch synthesis insinks and sources,” In Photoassimilate distribution in plants and crops:source-sink relationships. Zamski, E., ed., Marcil Dekker Inc. pp.139-168). AGP catalyzes what is viewed as the initial step in the starchbiosynthetic pathway with the product of the reaction being theactivated glucosyl donor, ADPglucose. This is utilized by starchsynthase for extension of the polysaccharide polymer (reviewed inHannah, L. Curtis [1996] “Starch synthesis in the maize endosperm,” In:Advances in Cellular and Molecular Biology of Plants, Vol. 4. B. A.Larkins and I. K. Vasil (eds.). Cellular and Molecular Biology of PlantSeed Development. Kluwer Academic Publishers, Dordrecht, TheNetherlands).

Initial studies with potato AGP showed that expression in E. coliyielded an enzyme with allosteric and kinetic properties very similar tothe native tuber enzyme (Iglesias, A., Barry, G. F., Meyer, C.,Bloksberg, L., Nakata, P., Greene, T., Laughlin, M. J., Okita, T. W.,Kishore, G. M., and Preiss, J. [1993] J. Biol Chem. 268:1081-86;Ballicora, M. A., Laughlin, M. J., Fu, Y., Okita, T. W., Barry, G. F.,and Preiss, J. [1995] Plant Physiol. 109:245-251). Greene et al.(Greene, T. W., Chantler, S. E., Kahn, M. L., Barry, G. F., Preiss, J.,and Okita, T. W. [1996] Proc. Natl. Acad. Sci. 93:1509-1513; Greene, T.W., Woodbury, R. L., and Okita, T. W. [1996] Plant Physiol.(112:1315-1320) showed the usefulness of the bacterial expression systemin their structure-function studies with the potato AGP. Multiplemutations important in mapping allosteric and substrate binding siteswere identified (Okita, T. W., Greene, T. W., Laughlin, M. J., Salamone,P., Woodbury, R., Choi, S., Ito, H., Kavakli, H., and Stephens, K.[1996] “Engineering Plant Starches by the Generation of Modified PlantBiosynthetic Enzymes,” In Engineering Crops for Industrial End Uses,Shewry, P. R., Napier, J. A., and Davis, P., eds., Portland Press Ltd.,London).

AGP enzymes have been isolated from both bacteria and plants. BacterialAGP consists of a homotetramer, while plant AGP from photosynthetic andnon-photosynthetic tissues is a heterotetramer composed of two differentsubunits. The plant enzyme is encoded by two different genes, with onesubunit being larger than the other. This feature has been noted in anumber of plants. The AGP subunits in spinach leaf have molecularweights of 54 kDa and 51 kDa, as estimated by SDS-PAGE. Both subunitsare immunoreactive with antibody raised against purified AGP fromspinach leaves (Copeland, L., J. Preiss (1981) Plant Physiol.68:996-1001; Morell, M., M. Bloon, V. Knowles, J. Preiss [1988] J. Bio.Chem. 263:633). Immunological analysis using antiserum prepared againstthe small and large subunits of spinach leaf showed that potato tuberAGP is also encoded by two genes (Okita et al., 1990, supra). The cDNAclones of the two subunits of potato tuber (50 and 51 kDa) have alsobeen isolated and sequenced (Muller-Rober, B. T., J. Kossmann, L. C.Hannah, L. Willmitzer, U. Sounewald [1990] Mol. Gen. Genet. 224:136-146;Nakata, P. A., T. W. Greene, J. M. Anderson, B. J. Smith-White, T. W.Okita, J. Preiss [1991] Plant Mol. Biol. 17:1089-1093). The largesubunit of potato tuber AGP is heat stable (Nakata et al. [1991],supra).

As Hannah and Nelson (Hannah, L. C., O. E. Nelson (1975) Plant Physiol.55:297-302; Hannah, L. C., and Nelson, Jr., O. E. [1976] Biochem. Genet.14:547-560) postulated, both Shrunken-2 (Sh2) (Bhave, M. R., S.Lawrence, C. Barton, L. C. Hannah [1990] Plant Cell 2:581-588) andBrittle-2 (Bt2) (Bae, J. M., M. Giroux, L. C. Hannah [1990] Maydica35:317-322) are structural genes of maize endosperm ADP-glucosepyrophosphorylase. Sh2 and Bt2 encode the large subunit and smallsubunit of the enzyme, respectively. From cDNA sequencing, Sh2 and Bt2proteins have predicted molecular weight of 57,179 Da (Shaw, J. R., L.C. Hannah [1992] Plant Physiol. 98:1214-1216) and 52,224 Da,respectively. The endosperm is the site of most starch deposition duringkernel development in maize. Sh2 and Bt2 maize endosperm mutants havegreatly reduced starch levels corresponding to deficient levels of AGPactivity. Mutations of either gene have been shown to reduce AGPactivity by about 95% (Tsai and Nelson, 1966, supra; Dickinson andPreiss, 1969, supra). Furthermore, it has been observed that enzymaticactivities increase with the dosage of functional wild type Sh2 and Bt2alleles, whereas mutant enzymes have altered kinetic properties. AGP isthe rate limiting step in starch biosynthesis in plants. Stark et al.placed a mutant form of E. coli AGP in potato tuber and obtained a 35%increase in starch content (Stark et al. [1992] Science 258:287).

The cloning and characterization of the genes encoding the AGP enzymesubunits have been reported for various plants. These include Sh2 cDNA(Bhave et al., 1990, supra), Sh2 genomic DNA (Shaw and Hannah, 1992,supra), and Bt2 cDNA (Bae et al., 1990, supra) from maize; small subunitcDNA (Anderson, J. M., J. Hnilo, R. Larson, T. W. Okita, M. Morell, J.Preiss [1989] J. Biol. Chem. 264:12238-12242) and genomic DNA (Anderson,J. M., R. Larson, D. Landencia, W. T. Kim, D. Morrow, T. W. Okita, J.Preiss [1991] Gene 97:199-205) from rice; and small and large subunitcDNAs from spinach leaf (Morell et al., 1988, supra) and potato tuber(Muller-Rober et al., 1990, supra; Nakata, P A., Greene, T. W.,Anderson, J. W., Smith-White, B. J., Okita, T. W., and Preiss, J. [1991]Plant Mol. Biol. 17:1089-1093). In addition, cDNA clones have beenisolated from wheat endosperm and leaf tissue (Olive, M. R., R. J.Ellis, W. W. Schuch [1989] Plant Physiol. Mol. Biol. 12:525-538) andArabidopsis thaliana leaf (Lin, T., Caspar, T., Sommerville, C. R., andPreiss, J. [1988] Plant Physiol. 88:1175-1181).

AGP functions as an allosteric enzyme in all tissues and organismsinvestigated to date. The allosteric properties of AGP were first shownto be important in E. coli. A glycogen-overproducing E. coli mutant wasisolated and the mutation mapped to the structural gene for AGP,designated as glyC. The mutant E. coli, known as glyC-16, was shown tobe more sensitive to the activator, fructose 1,6 bisphosphate, and lesssensitive to the inhibitor, cAMP (Preiss, J. [1984] Ann. Rev. Microbiol.419-458). Although plant AGP's are also allosteric, they respond todifferent effector molecules than bacterial AGP's. In plants,3-phosphoglyceric acid (3-PGA) functions as an activator while phosphate(PO₄) serves as an inhibitor (Dickinson and Preiss, 1969, supra).

Using an in vivo mutagenesis system created by the Ac-mediated excisionof a Ds transposable element fortuitously located close to a knownactivator binding site, Giroux et al. (Giroux, M. J., Shaw, J., Barry,G., Cobb, G. B., Greene, T., Okita, T. W., and Hannah, L. C. [1996]Proc. Natl. Acad. Sci. 93:5824-5829) were able to generate site-specificmutants in a functionally important region of maize endosperm AGP. Onemutant, Rev 6, contained a tyrosine-serine insert in the large subunitof AGP and conditioned a 11-18% increase in seed weight. In addition,published international application WO 01/64928 teaches that variouscharacteristics, such as seed number, plant biomass, Harvest Index etc.,can be increased in plants transformed with a polynucleotide encoding alarge subunit of maize AGP containing the Rev6 mutation.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to materials and methods useful forimproving crop yields in plants, such as those plants that producecereal crops. In one embodiment, the subject invention provides heatstable AGP enzymes and nucleotide sequences which encode these enzymes.In a preferred embodiment, the heat stable enzymes of the invention canbe used to provide plants having greater tolerance to highertemperatures, thus enhancing the crop yields from these plants. In aparticularly preferred embodiment, the improved plant is a cereal.Cereals to which this invention applies include, for example, maize,wheat, rice, and barley.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows heat stable maize endosperm AGP large subunit mutants.Percentage of AGP activity remaining after five minutes of heattreatment at 60° C. is shown.

FIG. 2 shows primary sequence alignment of the region surrounding HS 33mutation (SEQ ID NO: 1) in the AGP large subunits for maize (SEQ ID NO:2), wheat (SEQ ID NO: 3), barley (SEQ ID NO: 4), and potato (SEQ ID NO:5). Conserved regions are boxed.

FIG. 3 shows primary sequence alignment of the region surrounding HS 40mutation (SEQ ID NO: 6) in the AGP large subunits for maize (SEQ ID NO:7), wheat (SEQ ID NO: 8), barley (SEQ ID NO: 9), and potato (SEQ ID NO:10). Conserved regions are boxed. Bolded aspartic acid residuecorresponds to D413A allosteric mutant of potato LS (Greene, T. W.,Woodbury, R. L., and Okita, T. W. [1996] Plant Physiol. (112:1315-1320).Spinach leaf AGP sequence (SEQ ID NO: 11) is the activator site 2peptide identified in 3-PGA analogue studies (Ball, K. and Preiss, J.[1994] J. Biol. Chem. 269:24706-24711). The labeled lysine residue isbolded.

FIGS. 4A and 4B show molecular characterization of TS48 (SEQ ID NO: 12)and TS60 (SEQ ID NO: 17), respectively. Genetic lesion of TS48 (SEQ IDNO: 12) and corresponding residues are in bold. The amino acid number isindicated above the Leu to Phe mutation of TS48 (SEQ ID NO: 12). Thelast line is a consensus sequence. The Leu residue is highly conserved.Genetic lesions of TS60 (SEQ ID NO: 17) and corresponding residues arein bold. The amino acid numbers are indicated above the Glu to Lys andAla to Val mutations of TS60 (SEQ ID NO: 17). Boxed residues correspondto the HS 33 mutation (SEQ ID NO: 1) previously identified and shown tobe important in heat stability of the maize endosperm AGP. The last lineis a consensus sequence.

FIGS. 5A and 5B show molecular characterization of RTS 48-2 (SEQ ID NO:27) and RTS 60-1 (SEQ ID NO: 32), respectively. Genetic lesion of RTS48-2 (SEQ ID NO: 27) and corresponding residues are in bold. The aminoacid number is indicated above the Ala to Val mutation of RTS 48-2 (SEQID NO: 27). The last line is a consensus sequence. Of significance, themutation identified in RTS 48-2 (SEQ ID NO: 27) maps to the identicalresidue found in the heat stable variant HS13. HS 13 contained an Ala toPro mutation at position 177. Genetic lesion of RTS 60-1 (SEQ ID NO: 32)and corresponding residues are in bold. The amino acid number isindicated above the Ala to Val mutation of RTS 60-1 (SEQ ID NO: 32). Thelast line is a consensus sequence.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is an amino acid sequence of a region of the large subunitof AGP in maize containing the HS 33 mutation as shown in FIG. 2.

SEQ ID NO. 2 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 2.

SEQ ID NO. 3 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 2.

SEQ ID NO. 4 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 2.

SEQ ID NO. 5 is an amino acid sequence of a region of the large subunitof AGP in potato as shown in FIG. 2.

SEQ ID NO. 6 is an amino acid sequence of a region of the large subunitof AGP in maize containing the HS40 mutation as shown in FIG. 3.

SEQ ID NO. 7 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 3.

SEQ ID NO. 8 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 3.

SEQ ID NO. 9 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 3.

SEQ ID NO. 10 is an amino acid sequence of a region of the large subunitof AGP in potato as shown in FIG. 3.

SEQ ID NO. 11 is an amino acid sequence of a region of the large subunitof AGP in spinach as shown in FIG. 3.

SEQ ID NO. 12 is an amino acid sequence of a region of the large subunitof AGP in maize containing the TS48 mutation as shown in FIG. 4A.

SEQ ID NO. 13 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 4A.

SEQ ID NO. 14 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 4A.

SEQ ID NO. 15 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 4A.

SEQ ID NO. 16 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 4A.

SEQ ID NO. 17 is an amino acid sequence of a region of the large subunitof AGP in maize containing the TS60 mutation as shown in FIG. 4B.

SEQ ID NO. 18 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 4B.

SEQ ID NO. 19 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 4B.

SEQ ID NO. 20 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 4B.

SEQ ID NO. 21 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 4B.

SEQ ID NO. 22 is an amino acid sequence of a region of the large subunitof AGP in maize containing the TS60 mutation as shown in FIG. 4B.

SEQ ID NO. 23 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 4B.

SEQ ID NO. 24 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 4B.

SEQ ID NO. 25 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 4B.

SEQ ID NO. 26 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 4B.

SEQ ID NO. 27 is an amino acid sequence of a region of the large subunitof AGP in maize containing the RTS48-2 mutation as shown in FIG. 5A.

SEQ ID NO. 28 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 5A.

SEQ ID NO. 29 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 5A.

SEQ ID NO. 30 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 5A.

SEQ ID NO. 31 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 5A.

SEQ ID NO. 32 is an amino acid sequence of a region of the large subunitof AGP in maize containing the RTS60-1 mutation as shown in FIG. 5B.

SEQ ID NO. 33 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 5B.

SEQ ID NO. 34 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 5B.

SEQ ID NO. 35 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 5B.

SEQ ID NO. 36 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 5B.

SEQ ID NO. 37 is an amino acid sequence of a mutant maize large subunitof AGP.

SEQ ID NO. 38 is an amino acid sequence of a mutant maize large subunitof AGP.

SEQ ID NO. 39 is an amino acid sequence of a mutant maize large subunitof AGP.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns novel mutant polynucleotide molecules,and the polypeptides encoded thereby, that confer increased yield inplants grown under conditions of heat stress relative to plants havingwild type genotype. In specific embodiments, the polynucleotidemolecules of the subject invention encode maize endosperm ADP glucosepyrophosphorylase (AGP) and soluble starch synthase (SSS) enzymeactivities. The mutant enzymes confer increased stability to heat stressconditions during seed and plant development in seeds and plant tissueexpressing the enzymes as compared with wild type enzyme activities.

In one embodiment, a polynucleotide of the present invention encodes amutant large subunit of maize AGP containing a histidine-to-tyrosineamino acid substitution in the sequence of the polypeptide. Thissubstitution occurs at amino acid residue number 333, according to theaccepted number of the amino acids in this protein (Shaw and Hannah,1992, supra). The position of this substitution can be readilyidentified by a person skilled in the art. A second mutation exemplifiedin the subject invention is a threonine-to-isoleucine substitution atposition number 460 of the large subunit of the maize AGP protein.

Also exemplified are mutants wherein the histidine at position 333 ofthe maize large subunit of AGP is replaced with a phenylalanine,methionine, or glycine. Additional exemplified maize AGP large subunitmutants conferring increased heat stability are shown below in Table 1.

TABLE 1 Mutant Amino Acid Change HS 13 Ala to Pro at position 177 HS 14Asp to His at position 400, and Val to Ile at position 454 HS 16 Arg toThr at position 104 HS 33 His to Tyr at position 333 HS 33F His to Pheat position 333 HS 33M His to Met at position 333 HS 33G His to Gly atposition 333 HS 39 His to Tyr at position 333 HS 40 His to Tyr atposition 333, and Thr to Ile at position 460 HS 47 Arg to Pro atposition 216, and His to Tyr at position 333 RTS 48-2 Ala to Val atposition 177 RTS 60-1 Ala to Val at position 396

Because of the homology of AGP polypeptides between various species ofplants (Smith-White and Preiss [1992] J. Mol. Evol. 34:449-464), theordinarily skilled artisan can readily determine the correspondingposition of the mutations for maize AGP exemplified herein in AGP fromplants other than maize. For example, FIGS. 2 and 3 show primarysequence alignment for the region around the maize HS 33 and HS 40mutations in wheat, barley, and potato. Thus, the present inventionencompasses polynucleotides that encode mutant AGP of plants other thanmaize, including, but not limited to, wheat, barley, and rice, thatconfers increased heat stability when expressed in the plant.

cDNA clones for the subunits of the maize endosperm AGP (SH2 and BT2)and an E. coli strain deficient in the endogenous bacterial AGP (glg C⁻)(AC70R1-504) have facilitated the establishment of a bacterialexpression system to study the maize endosperm AGP. Expression of asingle subunit is unable to complement the glg C⁻ mutant, and noglycogen is produced (Iglesias, A., Barry, G. F., Meyer, C., Bloksberg,L., Nakata, P., Greene, T., Laughlin, M. J., Okita, T. W., Kishore, G.M., and Preiss, J. [1993] J. Biol Chem. 268: 1081-86). However,expression of both the large and small subunits on compatible expressionvectors fully complements the glg C⁻ mutation and restores glycogenproduction as evidenced by a dark, reddish-brown staining of coloniesexposed to iodine. Thus, complementation is easily identified by simplyexposing the colonies to iodine.

In one embodiment, E. coli glg C⁻ cells expressing the structural genesfor either potato or maize endosperm AGP were used. Cells containingpotato AGP genes can synthesize copious levels of glycogen when grown at37° C. or at 42° C. However, cells expressing maize endosperm AGP onlysynthesize glycogen at 37° C. This result demonstrates the heatsensitivity of wild-type maize endosperm AGP. That there is a differencebetween potato and maize AGP's in this regard provides an efficientsystem for screening for mutant cells that have heat stable variants ofthe maize endosperm AGP.

One aspect of the subject invention pertains to the efficientidentification of AGP which is heat stable. Accordingly, a plasmidcomprising a polynucleotide coding for the SH2 subunit of maize AGP waschemically mutagenized, as described below, placed into mutant E. colicells expressing the BT2 subunit, and the cells grown at 42° C. toselect for mutants that could produce glycogen at that temperature.Other mutagens known in the art can also be used. Eleven heritable,iodine staining mutants, termed heat stable (HS) mutants, were isolated.Crude extracts of these mutants were prepared and the heat stability ofthe resulting AGP was monitored. The mutants retained between 8-59% oftheir activity after incubation at 60° C. for five minutes (FIG. 1).This compares to the 1-4% routinely observed for wild-type AGP at thistemperature.

The results show that heat stable forms of enzymes can be createdaccording to the subject invention by mutation. Thus, one aspect of theinvention pertains to processes for producing and identifyingpolynucleotides encoding mutant starch biosynthesis enzymes havingincreased heat stability compared to wild type enzymes. Unexpectedly,total activity of the maize endosperm AGP before heat treatment waselevated about two- to three-fold in the majority of these mutants. Thissurprising result makes these mutants particularly advantageous for usein agriculture. Mutagenesis techniques as described herein can be usedaccording to the subject invention to identify other genes encoding heatstable starch biosynthesis enzymes.

The genes encoding several of the heat stable mutants exemplifiedherein, including two of the most heat stable HS mutants, HS 33 and HS40, were completely sequenced. HS 33, which retains 59% of its activityafter heat treatment, contains a single base pair mutation that changesa histidine residue at position 333 of the amino acid sequence of thepolypeptide to a tyrosine (FIG. 2). Primary sequence alignments with thelarge subunits from wheat and barley AGPs show that a histidine is alsopresent at the analogous residue (FIG. 3) (Ainsworth, C., Hosein, F.,Tarvis, M., Weir, F., Burrell, M., Devos, K. M., Gale, M. D. [1995]Planta 197:1-10). Sequence analysis of HS 40, which retains 41% of itsactivity post heat treatment, also contained a histidine to tyrosinemutation at position 333. An additional point mutation was identifiedthat generated a threonine to isoleucine substitution. The threonineresidue is highly conserved in AGP large subunits, while in AGP smallsubunits the analogous residue is either a cysteine or serine (Ainsworthet al., 1995, supra). The threonine to isoleucine substitution islocated close to the carboxyl terminus of the large subunit, and closeto a known binding site for the activator 3-PGA (FIG. 3).

Another aspect of the present invention pertains to mutant starchbiosynthesis enzymes, such as AGP, and the polynucleotides that encodethem, wherein these mutant enzymes are isolated by selecting fortemperature sensitive (TS) mutants which are then mutagenized andscreened for revertants that show enhanced stability. A further aspectof the invention concerns the methods for producing and identifying thepolynucleotides and mutant enzymes encoded thereby.

The subject invention also concerns heat stable mutants of AGP that havemutations in the small subunit of the enzyme. Also encompassed withinthe scope of the invention are polynucleotides that encode the mutantsmall subunits of AGP. Mutations in the small subunit of AGP that conferheat stability to the enzyme can also be readily prepared and identifiedusing the methods of the subject invention.

Plants and plant tissue bred to contain or transformed with the mutantpolynucleotides of the invention, and expressing the polypeptidesencoded by the polynucleotides, are also contemplated by the presentinvention. Plants and plant tissue expressing the mutant polynucleotidesproduce tissues that have, for example, lower heat-induced loss inweight or yield when subjected to heat stress during development. Plantswithin the scope of the present invention include monocotyledonousplants, such as rice, wheat, barley, oats, sorghum, maize, lilies, andmillet, and dicotyledonous plants, such as peas, alfalfa, chickpea,chicory, clover, kale, lentil, prairie grass, soybean, tobacco, potato,sweet potato, radish, cabbage, rape, apple trees, and lettuce. In aparticularly preferred embodiment, the plant is a cereal. Cereals towhich this invention applies include, for example, maize, wheat, rice,barley, oats, rye, and millet.

Plants having mutant polynucleotides of the invention can be grown fromseeds that comprise a mutant gene in their genome. In addition,techniques for transforming plants with a gene are known in the art.

Because of the degeneracy of the genetic code, a variety of differentpolynucleotide sequences can encode each of the variant AGP polypeptidesdisclosed herein. In addition, it is well within the skill of a persontrained in the art to create alternative polynucleotide sequencesencoding the same, or essentially the same, polypeptides of the subjectinvention. These variant or alternative polynucleotide sequences arewithin the scope of the subject invention. As used herein, references to“essentially the same” sequence refers to sequences which encode aminoacid substitutions, deletions, additions, or insertions which do notmaterially alter the functional activity of the polypeptide encoded bythe AGP mutant polynucleotide described herein.

As used herein, the terms “nucleic acid” and “polynucleotide sequence”refer to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited, wouldencompass known analogs of natural nucleotides that can function in asimilar manner as naturally-occurring nucleotides. The polynucleotidesequences include both the DNA strand sequence that is transcribed intoRNA and the RNA sequence that is translated into protein. Thepolynucleotide sequences include both full-length sequences as well asshorter sequences derived from the full-length sequences. It isunderstood that a particular polynucleotide sequence includes thedegenerate codons of the native sequence or sequences which may beintroduced to provide codon preference in a specific host cell. Allelicvariations of the exemplified sequences also come within the scope ofthe subject invention. The polynucleotide sequences falling within thescope of the subject invention further include sequences whichspecifically hybridize with the exemplified sequences. Thepolynucleotide includes both the sense and antisense strands as eitherindividual strands or in the duplex.

Substitution of amino acids other than those specifically exemplified inthe mutants disclosed herein are also contemplated within the scope ofthe present invention. Amino acids can be placed in the followingclasses: non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby a mutant AGP polypeptide having an amino acid ofone class is replaced with another amino acid of the same class fallwithin the scope of the subject invention so long as the mutant AGPpolypeptide having the substitution still retains increased heatstability relative to a wild type polypeptide. Table 2 below provides alisting of examples of amino acids belonging to each class.

TABLE 2 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, HisFor example, substitution of the tyrosine at position 333 in the HS 33,HS 39, HS 40 and HS 47 mutant maize endosperm AGP with other aminoacids, such as Glycine, Serine, Threonine, Cysteine, Asparagine, andGlutamine, are encompassed within the scope of the invention. Amino acidsubstitutions at positions other than the site of the heat stablemutation are also contemplated within the scope of the invention so longas the polypeptide retains increased heat stability relative to wildtype polypeptides.

The subject invention also concerns polynucleotides which encodefragments of the full length mutant polypeptide, so long as thosefragments retain substantially the same functional activity as fulllength polypeptide. The fragments of mutant AGP polypeptide encoded bythese polynucleotides are also within the scope of the presentinvention.

The subject invention also contemplates those polynucleotide moleculesencoding starch biosynthesis enzymes having sequences which aresufficiently homologous with the wild type sequence so as to permithybridization with that sequence under standard high-stringencyconditions. Such hybridization conditions are conventional in the art(see, e.g., Maniatis, T., E. F. Fritsch, J. Sambrook [1989] MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

The polynucleotide molecules of the subject invention can be used totransform plants to express the mutant heat stable enzyme in thoseplants. In addition, the polynucleotides of the subject invention can beused to express the recombinant variant enzyme. They can also be used asa probe to detect related enzymes. The polynucleotides can also be usedas DNA sizing standards.

The polynucleotide molecules of the subject invention also include thosepolynucleotides that encode starch biosynthesis enzymes, such as AGPenzymes, that contain mutations that can confer increased seed weight,in addition to enhanced heat stability, to a plant expressing thesemutants. The combination of a heat stabilizing mutation, such as forexample HS 33 or HS 40, with a mutation conferring increased seedweight, e.g., Rev 6, in a polynucleotide that encodes the large subunitof maize AGP is specifically contemplated in the present invention. U.S.Pat. Nos. 5,589,618 and 5,650,557 disclose polynucleotides (e.g., Rev6)that encode mutations in the large subunit of AGP that confer increasedseed weight in plants that express the mutant polypeptide.

Mutations in the AGP subunits that confer heat stability can be combinedaccording to the subject invention with phosphate insensitive mutants ofmaize, such as the Rev6 mutation, to enhance the stability of the Rev6encoded large subunit.

It is expected that enzymic activity of SSS will be impaired at highertemperatures as observed with AGP. Thus, mutagenized forms of SSS can beexpressed under increased thermal conditions (42° C.), to isolate heatstable variants in accordance with the methods described herein. Theseheat stable mutagenized forms of SSS, and the polynucleotides thatencode them, are further aspects of the subject invention.

The subject invention also concerns methods for increasing yieldcharacteristics of plants under conditions of heat stress byincorporating a polynucleotide of the present invention that comprises amutation in a starch biosynthesis enzyme that confers increasedstability or resistance to heat stress conditions and a mutation thatconfers increased yield characteristics on the plant. Increased yieldcharacteristics include, for example, increased seed number, increasedseed weight, increased plant biomass, and increased Harvest Index.

The subject invention also concerns methods for producing andidentifying polynucleotides and polypeptides contemplated within thescope of the invention. In one embodiment, gene mutation, followed byselection using a bacterial expression system, can be used to isolatepolynucleotide molecules that encode enzymes that can alleviateheat-induced loss in starch synthesis in plants. In a specificembodiment, a method for identifying a polynucleotide encoding a mutantstarch biosynthesis protein wherein the mutant starch biosynthesisprotein exhibits increased heat stability relative to a wild typeprotein comprises mutating a polynucleotide encoding a starchbiosynthesis protein, expressing the mutated polynucleotide in a cell toproduce a mutant starch biosynthesis protein, and determining whetherthe mutant starch biosynthesis protein exhibits increased heat stabilityrelative to the wild type starch biosynthesis protein.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are hereby incorporated byreference in their entirety to the extent they are not inconsistent withthe explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Use of Mutagenesis to Obtain Maize Endosperm AGP Heat StableVariants

The chemical mutagen hydroxylamine-HCl was initially used for the randommutagenesis of the large subunit expression plasmid. Hydroxylaminepreferentially hydroxylates the amino nitrogen at the C-4 position ofcytosine, and leads to a GC to AT transition (Suzuki, D. T., Griffith,A. J. F., Miller, J. H., and Lewontin, R. C. [1989] In Introduction togenetic analysis, Freeman, NY, 4th ed., pp. 475-499). The chemicalmutagen was chosen for its high mutation frequency. Limitations of thechemical mutagen are recognized, and if a large variety of geneticvariants are not isolated, PCR based random mutagenesis can beperformed. PCR mutagenesis generates a broader spectrum of mutationsthat include similar frequencies of transitions and transversion, andprovides an excellent alternative to the chemical method. The methodoutlined by Cadwell and Joyce (Cadwell, R. C. and Joyce, G. F. [1992]PCR Methods and Applications 2:28-33) can be followed for the PCR basedmethod.

Since the complete expression plasmid is used in the random mutagenesis,it is possible that mutations will occur outside of the coding region.Although it is expected that such mutations will not have any effect onthe heat stability of the maize endosperm AGP, each variant can besubcloned into an unmutated expression plasmid before any additionalcharacterization at the enzyme level is conducted. Both the large andsmall subunit expression plasmids can be constructed so that a NcoI/SacIdigestion will liberate the complete coding region. This can easily becloned back into a unmutated NcoI/SacI digested expression plasmid.

EXAMPLE 2 Molecular Characterization and Analysis of Heat Stable AGPVariants

Initially, 11 heat stable variants of the maize endosperm large subunitwere obtained. Sequencing was performed using DuPont and ABIinstrumentation. Sequence data can be routinely compared to theprogenitor wild-type allele. This analysis reveals the extent ofdiversity of changes conditioning heat stability.

Several of the sequenced HS mutants contained the identical histidine totyrosine change at amino acid position 333 in the large subunit.PCR-derived HS mutants can be quickly screened for the histidine totyrosine alteration by use of site-specific mutagenesis using primersthat change the tyrosine back to histidine.

EXAMPLE 3 Expression, Purification, and Kinetic Analysis of GeneticVariants

Conditions for the expression of the wild-type maize endosperm AGP in E.coli have been fully characterized. Optimum growth and inductionconditions vary somewhat from those previously published for potato AGPexpressed in E. coli (Iglesias et al., 1993, supra; Ballicora et al.,1995, supra). Induction at room temperature for 12-14 hrs in thepresence of 0.3 mM IPTG and 25 μg/ml nalidixic acid consistently giveshigh levels of expression and activity. Addition of 30% ammonium sulfateand 10 mM KH₂PO₄ ⁻/K₂HPO₄ ⁻ to the extraction buffer stabilizes themaize AGP in the crude extract.

Ammonium sulfate concentrated AGP is further purified by HydrophobicInteraction Chromatography using Tentacle C3 aminopropyl media (EMSeparations) packed into a Pharmacia HR 10/10 column. Protein binds tothe column in a buffer containing 1 M ammonium sulfate. AGP is elutedfrom the column by successive step gradient washes of buffer thatcontains 0.75 M, 0.5 M, 0.25 M, and 0 M ammonium sulfate. Wild-typemaize endosperm AGP typically elutes in the 0.25 M wash. C3 purifiedmaize endosperm AGP is further purified by anion exchange chromatographyusing Macro-Prep DEAE (BioRad) anion exchange media packed into aPharmacia HR 10/10 column. AGP is eluted by a linear gradient of 100-500mM KCl, and typically elutes at a salt concentration around 300 mM. APharmacia FPLC system is used for all chromatography steps. Theconditions for the individual purification steps are fullycharacterized. AGP activity during the purification is monitored by thepyrophosphorylysis assay, and purification steps are monitored bySDS-PAGE, Coomassie staining, and Western analysis using polyclonalantibodies specific to the maize endosperm AGP large and small subunits.

EXAMPLE 4 Enhanced Subunit Interaction

A totally unexpected pleiotropic effect of the HS maize endosperm AGPmutants is a two- to three-fold elevation of activity before heattreatment. One possible explanation for this result is that we have, bymutational change, shifted the ratio of SH2 and BT2 monomers andpolymers existing within the E. coli cell. Perhaps, in wild-type, only10% or less of the total proteins exist in the active heterotetramericform whereas in the mutants, this percentage is much higher. If thepolymer is more heat resistant than are the monomers, then the phenotypeof the mutants would be identical to what has been observed. Kineticanalysis can be used to determine changes in affinities for substratesand/or allosteric effectors.

To test the idea that the monomer/polymer ratio may be altered in thesemutants, the amounts of monomers and polymers in wild-type and inselected mutants both before and after heat treatment can be monitored.The availability of antibodies (Giroux, M. J. and Hannah, L. C. [1994]Mol. Gen. Genetics 243:400-408) for both subunits makes this approachfeasible. This can be examined both through sucrose gradientultracentrifugation and through gel chromatography and will readilydetermine which method is most efficient and definitive.

Since the higher plant AGP consists of two similar but distinct subunitsthat oligomerize to form the native heterotetrameric structure,mutations that enhance this interaction can provide added stability tothe enzyme. A yeast two-hybrid system (CLONTECH Laboratories, Palo Alto,Calif.) can be used to evaluate subunit interactions. Specific primersfor the amplification of the coding regions can be constructed. Theseprimers add unique restriction sites to the 5′- and 3′-ends so thatcloning facilitates the translational fusion of the individual subunitto the GAL4 DNA binding domain (pGBT9) or GAL4 activation domain(pGAD424). If the proteins cloned into the vectors interact, the DNAbinding domain and the activation domain will form a functionaltranscription activator. This in turn activates expression of thereporter gene, lac Z, cloned behind a GAL4 promoter.

Initially, conditions can be characterized with the wild-type subunits.The coding regions of the wild-type large and small subunits can becloned into the pGBT9 and pGAD424 yeast expression vectors. All possiblecombinations can be generated and tested. pGBT9 and pGAD424 vectorscontaining Sh2 and Bt2 can be cotransformed into the same yeast strain,and selected for growth on media lacking tryptophan (pGBT9) and leucine(pGAD424). Subunit interaction as a function of lacZ expression can bedetected two ways. Positive colonies are visually identified by aB-galactosidase filter assay. With this assay colonies are bound to thefilter, lysed, and incubated with an X-gal solution. Colonies thatexhibit a blue color can be analyzed. Subunit interaction can be furtheranalyzed by an enzyme assay specific for B-galactosidase. This allowsthe quantification of the interaction. Mutations that enhance subunitinteractions will give higher levels of B-galactosidase activity whenassayed.

EXAMPLE 5 Further Enhancement of Stability

The large subunit mutants isolated vary in their heat stabilitycharacteristics, suggesting the possibility of multiple mutations. Whilesequence analysis of mutants HS 33 and HS 40 reveal that the mutantsequences are not identical, both mutants contained the identicalhistidine to tyrosine change. Given the identification of different HSalterations within the SH2 protein, it is possible to efficientlypyramid these changes into one protein. Furthermore, any HS mutationswithin the small subunit can be co-expressed with HS SH2 mutants tofurther enhance the stability of the maize endosperm enzyme.

Multiple HS mutants within one subunit can easily be combined. Forexample, different unique restriction sites that divide the codingregions of Sh2 into three distinct fragments can be used. Whereappropriate, mutation combinations can be generated by subcloning thecorresponding fragment containing the added mutation. If two mutationsare in close proximity, then site-directed mutagenesis can be used toengineer such combinations. One method for site specific mutationsinvolves PCR, mutagenic primer, and the use of DpnI restrictionendonuclease. Primers can be constructed to contain the mutation in the5′ end, and used to PCR amplify using the proofreading polymerase Vent.Amplified DNA can then be digested with DpnI. Parental DNA isolated fromE. coli is methylated and hence susceptible to DpnI. Digested DNA issize fractionated by gel electrophoresis, ligated, and cloned into theexpression vectors. Mutations are confirmed by sequence analysis andtransformed into the AC70R1-504 strain carrying the wild-type smallsubunit. Combinatorial mutants can then be analyzed.

EXAMPLE 6 Identification of Additional Mutants at Position 333 of theLarge Subunit of Maize AGP

Hydroxylamine-HCl mutagenesis gives rise only to cytosine to thyminechanges, thereby limiting the types of possible substitutions. Becauseboth strands of DNA undergo mutagenesis, thymine to cytosine changesalso occur; however, taken together, only two of the 12 possible singlebase changes occur. Hence, not all possible amino acid substitutionswould have been produced by hydroxylamine-HCl mutagenesis.

Therefore, in order to prepare mutants where each of the 20 differentamino acids were inserted, individually, at position 333 of the largesubunit of maize endosperm AGP, a two step process was employed.Methodologies were derived basically from those of Stratagene. First,the codon encoding amino acid 333, plus the first base of the codon foramino acid 334, were removed via PCR-based site-specific mutagenesis(Suzuki et al., 1989, supra). Following screening for inactivity byiodine staining and subsequent sequencing to verify the deletion, theresulting plasmid was PCR mutagenized using a primer containingrandomized bases at the 333 codon plus the replacement base at the firstbase of the codon for amino acid 334. Resulting plasmids weretransformed into Bt2-containing E. coli mutant cells, screened viaiodine staining for activity at 37° C. and at 42° C., and subsequentlysequenced. Primers of lesser degeneracy were used in latter stages tomore efficiently generate codons not obtained in the first round.

All 20 amino acid substitutions were isolated following mutagenesis andall gave rise to staining at 37° C. Mutants were also scored for iodinestaining at the elevated temperature of 42° C. Coded strains were usedfor screening to remove any possible bias on the part of theinvestigators. Those mutants giving rise to staining equal to or greaterthan wildtype when grown at 42° C., are listed in Table 3 below.

As expected, the screening identified active enzymes having at position333 of the protein the wild type amino acid, i.e., histidine, or theamino acid of the HS 33 mutant, i.e., tyrosine. Phenylalanine, whichdiffers from tyrosine only by the absence of a polar hydroxyl group, wasalso identified. The screen also identified active enzymes having aminoacids differing substantially from tyrosine and phenylalanine, such as,for example, glycine.

AGP activities before and after treatment of freshly extracted enzymepreparations at 65° C. for 5 minutes were also measured and results areshown in Table 3. Of the eight amino acids selected through positivestaining E. coli plates grown at 42° C., three mutants (HS 33, HS 33F,and HS 33M having tyrosine, phenylalanine, or methionine at position333, respectively) proved superior activity in enzyme assays followingheat treatament at 65° C. While AGP activities from phenylalanine- andmethionine-containing AGPs are somewhat higher than that conditioned bythe tyrosine substitution, the differences in activity between thesethree preparations are small.

TABLE 3 Amino Acid Before Heat* After Heat* histidine (wt) 61 42tyrosine (HS 33) 100 77 phenylalanine 138 85 methionine 160 89 cysteine73 7 lysine 48 38 glycine 102 34 glutamine 65 21 *AGP activity in crudepreps is expressed as a percentage of HS 33 activity before heattreatment.

EXAMPLE 7 Combination of Heat Stability Mutations with Rev6

According to the subject invention, the heat stable mutations can becombined with a mutation associated with increased seed weight, such as,for example, the Rev6 mutation. The goal is to maintain the desiredphosphate insensitivity characteristic of Rev6 while enhancing itsstability. Rev 6/HS double mutants can be constructed and confirmed asdescribed herein. Double mutants can be transformed into AC70R1-504carrying the wild-type small subunit. Increased heat stability can beeasily identified by a positive glycogen staining on a low glucosemedia. Rev6 does not stain when grown on this media. Initially allmutant combinations can be screened enzymatically for maintenance ofphosphate insensitivity, and only combinations that maintain phosphateinsensitivity are further analyzed.

EXAMPLE 8 Cloning of SSS I Mutants

A glg A⁻ E. coli strain deficient in the endogenous bacterial glycogensynthase can be obtained from the E. coli Stock Center. Bacterialexpression vectors currently used for the expression of AGP can be usedfor expression of SSS.

One cloning strategy, as used, for example, with Sh2 and Bt2 (Giroux etal., 1996, supra), is the following: One primer contains a uniquerestriction plus the 5′ terminus of the transcript while the otherprimer contains another unique restriction site and sequences 3′ to thetranslational termination codon of the gene under investigation.Subsequent cloning of these gives rise to a translational fusion withinthe plasmid. These gene specific primers are initially used in RT-PCRreactions using poly A+RNA from developing endosperms.

Expression of the maize endosperm SSS I will complement the lack ofglycogen synthase activity in the glg A⁻ strain. Complementation shouldbe easily visualized with iodine staining as it is with the expressionof AGP in the glg C⁻ strain. Crude extracts can be incubated at varioustemperatures and lengths of time to determine the heat stability of SSSI. The glg A⁻ strain expressing the maize endosperm SSS I can be grownat various temperatures to determine if function is temperaturesensitive as it is with the AGP bacterial expression system. Once arestrictive temperature is established, a random mutagenesis can beconducted with the SSS I clone. Mutant forms of SSS I can be transformedinto the glg A⁻ strain, grown at the restrictive temperature, and heatstable variants identified by their ability to produce iodine-stainingglycogen at the restriction temperature.

EXAMPLE 9 Temperature Sensitive Mutants of Maize Endosperm ADP-GlucosePyrophosphorylase

As an alternative approach to identify additional variants withincreased stability, a reverse-genetics approach was employed.Temperature sensitive (TS) mutants have been isolated. These mutantsexhibit a negative iodine staining phenotype at 30° C. indicating a lackof function with the maize endosperm AGP. In contrast, when the mutantsare grown at 37° C. they can fully complement the mutation in thebacterial AGP. This clearly shows that the mutant AGPs are functional,and that the loss of function is temperature dependent. Wild type AGPexhibits a positive glycogen staining phenotype at 30° and 37° C. Thetemperature sensitive mutants were then used to produce second siterevertants that encode mutant AGP having enhanced stability.

Mutagenesis. pSh2 DNA was subjected to hydroxylamine mutagenesis(Greene, T. W., Chantler, S. E., Kahn, M. L., Barry, G. F., Preiss, J.,and Okita, T. W. [1996] Proc. Natl. Acad. Sci. 93:1509-1513) andtransformed into AC70R1-504 E. coli cells carrying the wild type pBt2small subunit plasmid. Cells were plated and grown at 30° C. Temperaturesensitive variants of AGP were identified by their negative iodinestaining phenotype at 30° C. Putative mutants were streaked again at 30°C. and 37° C. along with the wild type AGP as a control. Six mutantsthat consistently gave a negative iodine phenotype at 30° C. and apositive iodine staining phenotype at 37° C. were isolated. Expressionof wild type Sh2 and Bt2 gave a positive iodine staining phenotype atboth temperatures.Characterization of TS48 and TS60. Plasmid DNA from two temperaturesensitive mutants, TS48 and TS60, was isolated and sequenced to identifythe genetic lesion. A single point mutation that generated thereplacement of leucine at amino acid position 426 with phenylalanine wasidentified (FIG. 4A). This residue and surrounding region is highlyconserved in the cereal endosperm large subunits (LS) (Smith-White andPreiss, 1992, supra). In TS60, two point mutations were identified thatgenerated a glutamic acid to lysine change at amino acid 324 and analanine to valine mutation at position 359 (FIG. 4B). Glu-324 is highlyconserved among the LS and small (SS) subunits of AGP (Smith-White andPreiss, 1992, supra). Ala-359 and the surrounding amino acids are alsohighly conserved among the AGP LS. Of significance, the two mutationidentified in TS60 flank the HS 33 mutation described herein. The HS 33mutation, which has the histidine to tyrosine substitution at position333, was shown to greatly enhance heat stability of the maize endospermAGP. That the mutations of TS60 are in close proximity to the HS 33mutation is additional evidence that this region of the protein isimportant for stability.Isolation of second-site revertants. Isolation of the temperaturesensitive mutants provides a selectable phenotype for isolatingadditional variants that enhance the stability of AGP. Additionalhydroxylamine mutagenesis was conducted with TS48 and TS60 DNA toisolate second-site revertants that restore a positive glycogen stainingphenotype at 30° C. Hydroxylamine was used because the chemistry of themutagenesis eliminates the possibility of a direct reversion of theprimary mutation identified in the TS48 and TS60 mutants. This forcesthe selection of second-site mutations that can restore stability inthese temperature sensitive mutants.

Three revertants were isolated for TS48 and the molecularcharacterization of one mutant, RTS 48-2, is shown (FIG. 5A). RTS 48-2contains an alanine to valine mutation at amino acid position 177 inaddition to the parental mutation identified in TS48. This residue andthe surrounding region are highly conserved. The RTS 48-2 mutationcorresponds to the identical site of the mutation identified in the heatstable mutant, HS 13. The alanine residue was mutated to a proline atposition 177 in HS 13. That these two mutations map to the same site issignificant. The RTS 48-2 and HS 13 mutants were selected based onincreased stability using completely different approaches, and thusthese two mutations identify this site to be important in the stabilityof AGP.

Five second-site revertants were isolated for TS60 and the sequenceanalysis of one, RTS 60-1, is shown (FIG. 5B). An alanine to valinemutation at amino acid 396 was identified. This residue is highlyconserved among the AGP LS, and it also maps close to a heat stablemutation identified in HS 14.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

1. An isolated polynucleotide comprising a nucleotide sequence encodinga mutant large subunit of a plant ADP-glucose pyrophosphorylase (AGP)protein, or a biologically-active fragment of said mutant protein,wherein when said mutant large subunit protein is expressed to form amutant ADP-glucose pyrophosphorylase enzyme, said mutant enzyme, or abiologically-active fragment of said mutant enzyme, exhibits increasedheat stability relative to wild type ADP-glucose pyrophosphorylaseenzyme, and wherein said mutant large subunit protein encoded by saidpolynucleotide comprises an amino acid mutation wherein the amino acidcorresponding to the histidine at position 333 in the amino acidsequence of a wild type large subunit of ADP-glucose pyrophosphorylasepolypeptide of maize is replaced by an amino acid that confers saidincreased heat stability on said mutant enzyme.
 2. The polynucleotideaccording to claim 1, wherein said amino acid corresponding to saidhistidine at position 333 is replaced by a glycine.
 3. Thepolynucleotide according to claim 1, wherein said amino acidcorresponding to said histidine at position 333 is replaced by aphenylalanine.
 4. The polynucleotide according to claim 1, wherein saidamino acid corresponding to said histidine at position 333 is replacedby a methionine.
 5. The polynucleotide according to claim 1, whereinsaid mutant large subunit protein encoded by said polynucleotide furthercomprises an amino acid mutation that confers increased seed weight to aplant expressing said polynucleotide.
 6. The polynucleotide according toclaim 5, wherein said polynucleotide comprises the Rev6 mutation.
 7. Thepolynucleotide according to claim 5, wherein said polynucleotide encodesa maize large subunit AGP enzyme wherein at least one serine residue isinserted between amino acids 494 and 495 of a wild type AGP enzyme largesubunit.
 8. The polynucleotide according to claim 5, wherein saidpolynucleotide encodes a maize large subunit AGP enzyme wherein theamino acid pair tyrosine:serine is inserted between amino acids 494 and495 of the wild type AGP enzyme subunit or said polynucleotide encodes amaize large subunit AGP enzyme wherein the amino acid pairserine:tyrosine is inserted between amino acids 495 and 496 of a wildtype AGP enzyme large subunit.
 9. A method for increasing resistance ofa plant to heat stress conditions, said method comprising incorporatinga polynucleotide comprising a nucleotide sequence encoding a mutantlarge subunit of a plant ADP-glucose pyrophosphorylase (AGP) protein, ora biologically-active fragment of said mutant protein, wherein when saidmutant large subunit protein is expressed to form a mutant ADP-glucosepyrophosphorylase enzyme, said mutant enzyme, or a biologically-activefragment of said mutant enzyme, exhibits increased heat stabilityrelative to wild type ADP-glucose pyrophosphorylase enzyme, and whereinsaid mutant large subunit protein encoded by said polynucleotidecomprises an amino acid mutation wherein the amino acid corresponding tothe histidine at position 333 in the amino acid sequence of a wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by an amino acid that confers said increased heat stability onsaid mutant enzyme into the genome of said plant and expressing theprotein encoded by said polynucleotide molecule.
 10. The methodaccording to claim 9, wherein said plant is a monocotyledonous plant.11. The method according to claim 10, wherein said monocotyledonousplant is selected from the group consisting of rice, wheat, barley,oats, sorghum, maize, lilies, and millet.
 12. The method according toclaim 9, wherein said plant is Zea mays.
 13. The method according toclaim 9, wherein said plant is a dicotyledonous plant.
 14. A plant,plant tissue, or plant seed comprising a polynucleotide comprising anucleotide sequence encoding a mutant large subunit of a plantADP-glucose pyrophosphorylase (AGP) protein, or a biologically-activefragment of said mutant protein, wherein when said mutant large subunitprotein is expressed to form a mutant ADP-glucose pyrophosphorylaseenzyme, said mutant enzyme, or a biologically-active fragment of saidmutant enzyme, exhibits increased heat stability relative to wild typeADP-glucose pyrophosphorylase enzyme, and wherein said mutant largesubunit protein encoded by said polynucleotide comprises an amino acidmutation wherein the amino acid corresponding to the histidine atposition 333 in the amino acid sequence of a wild type large subunit ofADP-glucose pyrophosphorylase polypeptide of maize is replaced by anamino acid that confers said increased heat stability on said mutantenzyme.
 15. The plant, plant tissue, or plant seed according to claim14, wherein said plant, plant tissue, or plant seed is monocotyledonous.16. The plant, plant tissue, or plant seed according to claim 15,wherein said monocotyledonous plant, plant tissue, or plant seed isselected from the group consisting of rice, wheat, barley, oats,sorghum, maize, lilies, and millet.
 17. The plant, plant tissue, orplant seed according to claim 14, wherein said plant is Zea mays or saidplant tissue or plant seed is from Zea mays.
 18. The plant, planttissue, or plant seed according to claim 14, wherein said plant, planttissue, or plant seed is dicotyledonous.
 19. The polynucleotideaccording to claim 2, wherein said mutant protein encoded by saidpolynucleotide comprises the amino acid sequence shown in SEQ ID NO. 37.20. The polynucleotide according to claim 3, wherein said mutant proteinencoded by said polynucleotide comprises the amino acid sequence shownin SEQ ID NO.
 38. 21. The polynucleotide according to claim 4, whereinsaid mutant protein encoded by said polynucleotide comprises the aminoacid sequence shown in SEQ ID NO. 39.